Bottom Line:
Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX).Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis.It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway.

ABSTRACTNon-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG2000-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.

f4-ijn-9-921: Confocal images of A549 cells treated with PEG-LP/DOX, GE11-LP/DOX, or GE11-LP/DOX preincubated with free GE11 for 1 hour or 2 hours.Notes: The nucleus was stained with Hoechst 33342 and visualized as blue fluorescence, and PEG-LP/DOX and GE11-LP/DOX was visualized as red fluorescence. These images were taken at a magnification of 40×.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes.

Mentions:
Confocal microscopy was used to investigate cellular uptake of the liposomes and the results were shown qualitatively by the fluorescent images in Figure 4. A549 cells treated with GE11-LP/DOX showed stronger fluorescence than PEG-LP/DOX at both 1 hour and 2 hours, suggesting that GE11 could facilitate effective cellular uptake of the liposome. To verify further the role of GE11, A549 cells were preincubated with free GE11, which is believed to block specific binding of the GE11-modified liposome. As shown in Figure 4C and F, the uptake of GE11-LP/DOX was markedly inhibited to a degree similar to that achieved by PEG-LP/DOX, indicating that GE11 indeed possessed specific targeting ability for A549 cells. In addition, it was of note that the fluorescence of GE11-LP/DOX and PEG-LP/DOX became stronger with prolongation of the culture time, indicating that cellular uptake of the liposomes occurred in a time-dependent manner.

f4-ijn-9-921: Confocal images of A549 cells treated with PEG-LP/DOX, GE11-LP/DOX, or GE11-LP/DOX preincubated with free GE11 for 1 hour or 2 hours.Notes: The nucleus was stained with Hoechst 33342 and visualized as blue fluorescence, and PEG-LP/DOX and GE11-LP/DOX was visualized as red fluorescence. These images were taken at a magnification of 40×.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes.

Mentions:
Confocal microscopy was used to investigate cellular uptake of the liposomes and the results were shown qualitatively by the fluorescent images in Figure 4. A549 cells treated with GE11-LP/DOX showed stronger fluorescence than PEG-LP/DOX at both 1 hour and 2 hours, suggesting that GE11 could facilitate effective cellular uptake of the liposome. To verify further the role of GE11, A549 cells were preincubated with free GE11, which is believed to block specific binding of the GE11-modified liposome. As shown in Figure 4C and F, the uptake of GE11-LP/DOX was markedly inhibited to a degree similar to that achieved by PEG-LP/DOX, indicating that GE11 indeed possessed specific targeting ability for A549 cells. In addition, it was of note that the fluorescence of GE11-LP/DOX and PEG-LP/DOX became stronger with prolongation of the culture time, indicating that cellular uptake of the liposomes occurred in a time-dependent manner.

Bottom Line:
Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX).Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis.It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway.

ABSTRACTNon-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG2000-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.